PROJECT SUMMARY The placenta plays a critical role in supporting fetal development. Consequently, placental dysfunction can have catastrophic consequences for both the fetus and the mother. While insight into the mechanisms underlying the placenta's role in supporting pregnancy is lacking, we know that certain microRNA (miRNA) species are central to this process. In this project, we focus on a family of primate-specific miRNAs that are expressed from the largest cluster of human miRNAs, located on chromosome 19 (called Ch. 19 miRNA cluster, or C19MC). These miRNAs are highly expressed, almost exclusively in the placenta, where they are implicated in the defense against viral infection. These C19MC miRNAs are also involved in regulation of trophoblast migration and invasion, which are critical for proper placentation. Indeed, deviant expression patterns of C19MC miRNA species have been reported in pregnancies complicated by placental insufficiency. Moreover, C19MC miRNAs are ectopically expressed in several forms of aggressive tumors, underscoring their role in controlling cell fate decisions and the need for tight control of C19MC expression. To decipher the mechanisms underlying the regulation of C19MC expression, we recently generated several transgenic mouse lines, engineered to genomically harbor a bacterial artificial chromosome (BAC) plasmid containing the entire human C19MC region. Although the C19MC is primate specific, we found that the expression pattern of these human miRNAs in mouse tissues is extraordinarily parallel to that observed in humans, implying that the BAC- based transgene contains all regulatory elements necessary for adequate transcriptional control of the C19MC, and that the mouse placenta provides an epigenomic context akin to the human placenta. We used ATAC-seq assay to map the open chromatin landscape of the C19MC region, and luciferase reporter assays to identify genomic DNA domains with enhancer activity. Our goal in this project is to analyze the function and the regulatory potential of these genomic elements, using a set of functional assays. We will first use the CRISPR- Cas9 system to genetically disable endogenous genomic elements in placental cell lines, and determine the consequences on C19MC expression. We will further analyze the functional importance of the C19MC regulatory elements in two different mouse models: one designed to interrogate the ability of each genomic element to activate a heterologous promoter in the mouse placenta, using lentivirus-mediated placenta-specific transgenesis; the other is our ?humanized? transgenic mouse model, carrying the C19MC locus, which will be used to assess the influence of each discrete element, in the context of native chromatin, on placental expression. Using these non-overlapping, complementary approaches, this project will provide critically needed information about the mechanisms underlying the unique expression of C19MC miRNAs and their placental specificity. This project will provide essential information on trophoblast invasion and differentiation, placental dysfunction, trophoblast protection against viral infections during pregnancy, and carcinogenesis.